Snapgene tool

JCat (http://www.mrc-lmb.cam.ac.uk/ms/methods/codon.html, accessed on 21 April 2021) was used for the reverse translation process and the optimization of codons to gain the highest expression in E. coli using computational algorithms. The highest expression was assured by the Jcat tool. The GC contents percentage and CAI scores of the immunogenic construct were recorded. It was then cloned into PET-61-DEST (+) plasmid. Appropriate restriction enzymes were added to cut the plasmid and the immunogen at the particular parts. The immunogen was performed by the SnapGene tool (GSL Biotech, Evanston, IL, USA).

The Java Codon Adaptation Tool or JCat server was employed (http://www.jcat.de/) for the purpose of in silico codon adaptation in model organism E. coli strain K12 for the expression of protein vaccine (Grote et al., 2005 (link)). Vaccine constructs were reverse transcribed to possible DNA sequence and filters were applied to avoid rho-independent transcription termination, prokaryotic ribosome binding sites and cleavage sites of various restriction enzymes (BamHI and XhoI). The reverse-transcribed DNA sequence (RT-DNA) thus obtained is conjugated with XhoI and BamHI restriction sites at N-terminal and C-terminal sites, respectively. Next this adapted DNA sequence is incorporated into the multiple cloning site (MCS) of pET-28a (+) vector using the SnapGene tool (GSL Biotech, 2020 ).